The proposed research will focus on biophysical studies of activation of oxygen with special emphasis on cytochrome oxidase as the archetypal oxygen activating system. We wish to understand how this protein affects the processes of activation of 02, electron transfer, and the coupling of electron transfer and proton translocation in respiration. Specific projects to be undertaken include the following: 1. The ligand/metal/protein dynamics of the photodissociation and rebinding of various ligands to cytochrome oxidase will be investigated. Spectroscopically active ligand binding probes including 02, CO, NO, CN-, and isocyanide derivatives will be investigated using a multitechnique spectroscopic and kinetics approach, including FTIR, transient UV-Vis, time-resolved magnetic circular dichroism, EPR, and X-ray absorption spectroscopy on timescales of nanoseconds to seconds. The intramolecular electron transfer in cytochrome oxidase will be studied following photolysis of partially reduced ligand-bound enzyme complexes. These studies will provide new insight into what factors control the reactivity of the active site of this protein and how the physical characteristics of the ligand (spin state, charge, and size) affect reactivity. 2. The direct reaction of 02 with the reduced unliganded cytochrome oxidase will be studied without the need for photodissociation of CO using a new superfast direct-mixing method, pulsed-accelerated-flow. An alternative novel approach to the study of this reaction, wherein dioxygen is produced in situ on any relevant timescale by photodissociation of synthetic dioxygen carriers, will be pursued. Dicobalt mu-peroxo and mu-superoxo complexes of selected polyamines will be explored for photoproduction of 02. The results from both approaches will be compared to previous flow-flash results (CO present). 3. The newly discovered nonheme metals in cytochrome oxidase, Zn, Mg, and Cux, will be investigated with respect to their structural and mechanistic roles in the physiological function of the enzyme by various chemical and spectroscopic techniques. Removal of the metals and replacement with spectroscopically active metals (CO2+) and luminescent lanthanides will be attempted to obtain information regarding the nature of the active sites. The location of the Mg binding site and the possibility of it serving as a nucleotide binding site will be explored using novel fluorescent photoaffinity analogs of ATP. A comprehensive study, including metal analyses and characterization by EPR, will be carried out to establish the exact stoichiometry and the nature of Cux.